PROJECT SUMMARY Centrioles are microtubule-organizing structures with two primary roles in cells: 1) They function as basal bodies that anchor cilia and 2) They are a major component of the centrosome, the primary microtubule organizing center that functions in the spindle apparatus during cell division and to organize the cytoskeleton. Due to their multi-functionality, defects in centriole structure and biogenesis lead to a variety of maladies from developmental defects like microcephaly to aggressive cancers. Centrioles are barrel shaped, radial symmetric arrays of triplet microtubules. They use hundreds of proteins to promote microtubule organization and to stabilize the centriole structure; maintenance of centriole structure is important since centrioles experience force during ciliary beating and DNA segregation. Proteomic studies identified hundreds of proteins that localize to mature centrioles, but the mechanisms that drive centriole assembly and stabilization are poorly understood. Understanding the biogenesis and structure of centrioles will illuminate how these elaborate macromolecular complexes function. Here, I propose to use the ciliate Tetrahymena as a model organism to study centriole assembly and structure. Tetrahymena are ideal for centriole study for several reasons including that their assembly occurs at well-defined locations, allowing for observation of early events in this process. Additionally, they have hundreds of centrioles per cell, making biochemical and structural biology approaches practical. One of the earliest steps in centriole assembly is the formation of the ?cartwheel?, which establishes the centriole's highly conserved nine-fold symmetry. The mechanisms that control cartwheel assembly are not known. In Aim 1, I will use super-resolution fluorescence microscopy to test models of cartwheel assembly. These models include that cartwheel proteins depend on one another for proper incorporation into the centriole and that a preexisting ?mother? centriole templates nascent cartwheels. This aim will define the mechanisms that control the earliest steps in centriole assembly. The general morphology of the centriole has been known since the 1950s, and recent cryo-tomography studies revealed the structures of the cartwheel and triplet microtubules at 30-40. In Aim 2, I will improve the resolution of centriole structure to less than 10 using new advances in cryo-tomography. This will reveal the structural interactions that form the overall centriole architecture, identify the location of proteins within the complex, and provide a structural framework for understanding how the centriole facilitates microtubule organization and stabilization from force.